Authors: MUHAMMAD SAJJAD IQBAL, SHAHID NADEEM, SHAHID MEHBOOB, ABDUL GHAFOOR, MUHAMMAD IBRAHIM RAJOKA, AFSARI SHARIF QURESHI, BUSHRA NIAZ
Abstract: Nigella sativa L. has industrial, cosmetic, and pharmaceutical uses but has not been adequately characterized in Pakistan. This investigation was carried out to explore genotype specific fingerprinting of 32 N. sativa L. genotypes based on randomly amplified polymorphic DNA markers. From 58 random primers used, 15 primers generated 249 reproducible and scorable amplification products across all the genotypes, out of which 164 (66%) fragments were polymorphic revealing a high level of polymorphism among these genotypes. The proportion of common bands was low (34%). The size of the amplification products on agarose gels ranged between 0.5 and 10.0 kb. In 13 genotypes, 27 bands of different masses (kilobases) were recorded and were considered specific to those genotypes. These specific/amplified PCR products can be used as molecular markers for identification of germplasm and resource protection of Nigella sativa L. genotypes. Specific bands were observed for individual primers that could resolve genetic diversity among several genotypes (PK-020545, PK-020567, PK-020576, PK-020585, PK-020592, PK-020620, PK-020631, PK-020646, PK-020663, PK-020729, PK-020742, PK-020749, and PK-020868). UPGMA cluster analysis indicated 7 distinct clusters, 1 (C-3) comprising 9 accessions of N. sativa L., while C-7, C-5, and C-6 included 7, 6, and 5 accessions, respectively. C-4 and C-2 included 2 accessions each. Cluster 1 remained distinct as it had only 1 accession (PK-020646), indicating its higher genetic diversity from all other species. The overall grouping pattern of clusters corresponded well with principal component analysis and confirmed overall patterns of genetic variability among the species. These genotypes could be used as parents for random mutagenesis, or incorporated for gene recombination studies before marker assisted selection/breeding can be used for crop improvement. Moreover, these DNA based markers can be suitable for genetic distance estimation because they detected potentially large number of polymorphisms. Future research is needed to identify molecular markers linked to important traits (yield and oil quality) and locating quantitative traits loci (QTL) for improvement of N. sativa L. germplasm.
Keywords: Biodiversity, genetic relatedness, genotype specific bands, PCR, polymorphism, RAPD, random priming, UPGMA
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